Many clinical and biological applications require that small particles (e.g., cells, multi-cellular organisms, micro-spheres, etc.) be sorted and dispensed. Small particles can be sorted and dispensed using a flow cytometer. In a typical flow cytometer, particles are introduced into a “sheath” fluid that flows into a vertical chamber or tube. The sheath fluid hydrodynamically focuses the particles toward the center of the chamber.
In some flow cytometers, the particles are electrostatically sorted. For example, in U.S. Pat. No. 6,248,590, the sheath fluid and suspended particles exit the chamber through a nozzle and free fall through open air. In free fall, the sheath fluid breaks up into droplets that contain the particles. The droplets pass through a sensing zone where the particles are interrogated by a sensor (e.g., optical sensor, etc.). Based on the results of the interrogation, the particles are sorted by diverting them (in either of two directions) or by not diverting them. The droplets are diverted by: (1) electrically charging them and (2) passing them through an electric field. The direction in which a charged droplet is diverted depends upon the charge (i.e., positive or negative) on the droplet. A neutral (i.e., uncharged) droplet falls un-diverted through the electric field. Vessels (e.g., multi-well plates, etc.) that are appropriately positioned below the chamber receive the diverted and un-diverted droplets.
In some other flow cytometers, the particles are pneumatically sorted. For example, in PCT Published Patent Application WO/00/11449, particles that are hydrodynamically-focused by the sheath fluid pass, one-by-one, through a sensing zone that is located within the vertical chamber. There, the particles are interrogated by a sensor. After interrogation, the sheath fluid and particles exit the chamber into open air. A desirable particle, as identified by the sensor and processing electronics, falls undisturbed (within a droplet) to an underlying receiver. In contrast, when undesirable particles are detected, an electrically-operated valve introduces a flow of compressed gas into the falling drops of sheath fluid thereby changing the path of the free-falling sheath fluid and undesirable particles. The diverted fluid and particles are collected in a waste reservoir and recycled, as appropriate.
These electrostatic and pneumatic flow cytometers suffer from a variety of drawbacks. One drawback is the need for very accurate timing. In particular, to sort particles, the particle-containing sheath fluid must be diverted—by electrostatics or a blast of gas—at a precise time after a particle is detected. Since the time delay is premised on a specific set of conditions (e.g., flow rate, temperature, flow pattern, etc.), these conditions must be maintained for accurate operation. Consequently, these systems require frequent re-calibration.
Another drawback of many prior art flow cytometers is that they are open systems. That is, the sheath fluid and particles are typically expelled into the ambient environment (e.g., air, etc.) before they are sorted. This approach results in a number of processing inefficiencies including slow response time, solvent loss due to evaporation and, to the extent that particles and/or associated moieties (e.g., chemical species attached to the particles, etc.) are oxygen sensitive, degradation.